Isolated pediococcus acidilactici 05b0111 and method of producing exopolysaccharide

ABSTRACT

Disclosed herein is an isolated  Pediococcus acidilactici  05B0111 capable of producing an exopolysaccharide. The  Pediococcus acidilactici  05B0111 is deposited in Biosource Collection and Research Center (BCRC) of Food Industry Research and Development Institute (FIRDI) under an accession number BCRC 910420 and deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under an accession number DSM 22345. A method of producing an exopolysaccharide is also disclosed. The method involves cultivating an isolated  Pediococcus acidilactici  in a suitable medium under condition such that the exopolysaccharide is formed. A pharmaceutical composition including the aforementioned  Pediococcus acidilactici  05B0111 and a food product including the aforementioned  Pediococcus acidilactici  05B0111 are disclosed as well.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of Taiwanese Application No. 098134914,filed on Oct. 15, 2009.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an isolated Pediococcus acidilactici 05B0111having high exopolysaccharide-producing ability. The Pediococcusacidilactici 05B0111 is deposited in Biosource Collection and ResearchCenter (BORG) of Food Industry Research and Development Institute(FIRDI) under an accession number BCRC 910420 and deposited in DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under anaccession number DSM 22345. This invention also relates to a method ofproducing an exopolysaccharide, which comprises cultivating an isolatedPediococcus acidilactici in a suitable medium under condition such thatthe exopolysaccharide is formed. This invention also relates to apharmaceutical composition containing the isolated Pediococcusacidilactici 05B0111 and to a food product containing the isolatedPediococcus acidilactici 05B0111.

2. Description of the Related Art

Exopolysaccharides (EPS) or extracellular polysaccharides (EPS) aremacromolecules produced by microorganisms and secreted outside the cellwall of the microorganisms. EPS are generally classified into (1)capsular EPS that form a slime layer loosely attached to a cell surfaceand (2) unattached EPS that can be secreted to an environment.

Lactic acid bacteria (LAB) are generally recognized as safe (GRAS), andare widely used probiotics. A number of LAB are able to produce EPS.Most of EPS-producing LAB belong to the genus Streptococcus, the genusLactobacillus, the genus Lactococcus, the genus Leuconostoc, or thegenus Pediococcus (Petronella J. Looijesteijn et al. (1999), Applied andEnvironmental Microbiology, 65:5003-5008; T. Smitinont et al. (1999),International Journal of Food Microbiology, 51:105-111; PatriciaRuas-Madiedo et al. (2007), Applied and Environmental Microbiology,73:4385-4388). In addition, some strains belonging to the genusBifidobacterium are also shown to have the ability to produce EPS (P.Ruas-Madiedo and C. G. de los Reyes-Gavilán (2005), J. Dairy Sci.,88:843-856).

Generally speaking, EPS generated by LAB can be categorized ashomopolysaccharides (HoPS) and heteropolysaccharides (HePS). HoPS arecomposed of a single type of monosaccharide. Examples of HoPS includeα-glucans (e.g., dextran), β-glucans, and fructans (e.g., levan-type andinulin-type fructans). HePS are composed of repeating units that includedifferent monosaccharides. The repeating units of HePS are normallycomposed of 3-8 monosaccharides, and mostly include D-glucose,D-galactose, and L-rhamnose. In a few cases, HePS includeN-acetylglucosamine, N-acetylgalactosamine, fucose, glucuronic acid, andnon-carbohydrate substituents (e.g., phosphate, acetyl group, andglycerol).

EPS produced by LAB are capable of enabling food to have specialrheological properties and texture, thereby frequently serving asviscosifiers, stabilizer, emulsifiers, and gelling agents in the foodindustry. Additionally, many good effects of EPS produced by LAB onhealth of hosts have been discovered, for example, reduction incholesterol, modulation of immune activity, antitumor, etc. Since EPSproduced by LAB are beneficial to bioactivity, the same has been widelyused.

However, EPS produced by bacteria are usually unstable, and yield of thesame is normally very low. Since EPS is in great demand, various factorsaffecting productivity of EPS of LAB have been investigated so as todevelop a new cultivation technology for increasing yield of EPS.Petronella J. Looijesteijn et al. mention that strains, cultureconditions, and medium composition influence the amount of microbial EPSproduced by a certain species, and that a type of a carbon sourcestrongly affects productivity of EPS and may influence composition ofEPS as well. Petronella J. Looijesteijn et al. further state thatLactobacillus delbrueckii subsp. bulgaricus NCFB 2772 generates threetimes more EPS using glucose than using fructose as a carbon source, andthat yields of EPS produced by Lactobacillus casei CG11, Lactobacillusrhamnosus C83, and Streptococcus salivarius subsp. Thermophilus areobviously affected by a carbon source (Petronella J. Looijesteijn et al.(1999), supra).

P. Ruas-Madiedo and C. G. de los Reyes-Gavilán mention that productionof EPS by Lactobacillus casei CRL87 is 1.7-fold higher in galactose thanin glucose, and that Lactococcus lactis subsp. Cremoris B40 produceslarger amounts of EPS in glucose than in fructose (P. Ruas-Madiedo andC. G. de los Reyes-Gavilán (2005), supra). T. Smitinont et al. havereported that two Pediococcus pentosaceus strains (AP-1 and AP-3)isolated from traditional Thai food are capable of producing EPS in highyield, and that in liquid media containing 2% sucrose as a carbonsource, AP-1 and AP-3 strains are able to respectively produce 6.0 g/LEPS and 2.5 g/L EPS (T. Smitinont et al. (1999), supra).

In addition, among known species belonging to the genus Pediococcus,Pediococcus damnosus, Pediococcus parvulus, and Pediococcus pentosaceusare able to produce EPS (Maite Due{hacek over (n)}as et al. (2003),International Journal of Food Microbiology, 87:113-120; S. Velasco atal. (2006), International Journal of Food Microbiology, 111: 252-258; T.Smitinont et al. (1999), supra). However, the applicants indicate thatnone of literatures or prior art has disclosed theexopolysaccharide-producing ability of Pediococcus acidilactici andapplications thereof.

During research, the applicants found that Pediococcus acidilactici hasexopolysaccharide-producing ability. Particularly, the applicants havescreened a Pediococcus acidilactici isolate, which is phylogeneticallydifferent from the published Pediococcus strains, from fermented foodproducts. The Pediococcus acidilactici isolate has greatexopolysaccharide-producing ability, thereby being expected to generatea large number of EPS.

SUMMARY OF THE INVENTION

Therefore, according to a first aspect, this invention provides anisolated Pediococcus acidilactici 05B0111 capable of producing anexopolysaccharide. The Pediococcus acidilactici 05B0111 is deposited inBiosource Collection and Research Center (BCRC) of Food IndustryResearch and Development Institute (FIRDI) under an accession numberBCRC 910420 and deposited in Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ) under an accession number DSM 22345.

According to a second aspect, this invention provides a method ofproducing an exopolysaccharide, which comprises cultivating an isolatedPediococcus acidilactici in a suitable medium under condition such thatthe exopolysaccharide is formed.

According to a third aspect, this invention provides a pharmaceuticalcomposition comprising the aforementioned isolated Pediococcusacidilactici 05B0111.

According to a fourth aspect, this invention provides a food productcomprising the aforementioned isolated Pediococcus acidilactici 05B0111.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become apparent in the following detailed description of thepreferred embodiments with reference to the accompanying drawings, ofwhich:

FIG. 1 shows a full-length nucleotide sequence of 16S rDNA ofPediococcus acidilactici 05E0111 according to this invention;

FIG. 2 is a bar diagram showing effects of different carbohydrates onexopolysaccharide-producing ability of Pediococcus acidilactici 05B0111according to this invention;

FIG. 3 is a bar diagram showing exopolysaccharide concentration inorange juice containing 10% sucrose at different time points (8 hrs and72 hrs) of cultivation. The orange juice containing 10% sucrose wasinoculated with Pediococcus acidilactici 05B0111 according to thisinvention, followed by cultivation at a respective one of 30° C. and 37°C.; and

FIG. 4 is a graph showing concentrations of TNF-α secreted by mouseBALB/c macrophages RAW 264.7 at different exopolysaccharideconcentrations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that, if any prior art publication is referred toherein, such reference does not constitute an admission that thepublication forms apart of the common general knowledge in the art, inTaiwan or any other country.

For the purpose of this specification, it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

Lactic acid bacteria (LAB) are generally recognized as safe (GRAS), andare widely used probiotics. Many beneficial effects of LAB on health ofhosts have been verified. Exopolysaccharides produced by LAB can bewidely used. In order to massively produce exopolysaccharides, theapplicants have attempted to look for a LAB isolate having highexopolysachharide-producing ability from various LAB.

Accordingly, the applicants preliminarily screened 26 LAB isolatescapable of producing exopolysaccharides from fermented food productsavailable in the market. The 26 LAB isolates were subjected to 16S rDNAsequence analysis, and the results show that 5 LAB isolates out of the26 LAB isolates phylogenetically belong to Pediococcus acidilactici.Particularly, by virtue of evaluation of exopolysaccharide-producingability and phylogenetic novelty, the applicants found that among theaforementioned 5 LAB isolates, Pediococcus acidilactici 05B0111 has thebest exopolysaccharide-producing ability and is regarded as a novel LABisolate. On Feb. 10, 2009, Pediococcus acidilactici 05B0111 wasdeposited in Biosource Collection and Research Center (BCRC) of FoodIndustry Research and Development Institute (FIRDI) under an accessionnumber BCRC 910420. Furthermore, on Mar. 5, 2009, Pediococcusacidilactici 05B0111 was deposited in Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ) under an accession numberDSM 22345 so as to meet the requirement of the Budapest Treaty.

Accordingly, this invention provides a method of producing anexopolysaccharide, which comprises cultivating an isolated Pediococcusacidilactici in a suitable medium under condition such that theexopolysaccharide is formed. Preferably, the isolated Pediococcusacidilactici cultivated in the method is the isolated Pediococcusacidilactici 05B0111. Ina preferred embodiment of this invention, theresultant exopolysaccharide is purified.

As used herein, the term “cultivation”, the term “culturing”, and theterm “fermentation” can be interchangeably used.

According to this invention, the medium suitable to cultivatePediococcus acidilactici 05B0111 can be a synthetic medium or an ediblematerial. Examples of the synthetic medium suitable for the presentinvention include, but are not limited to, MRS broth. Examples of theedible material suitable for the present invention include, but are notlimited to, a fluid milk product (e.g., milk and concentrated milk),milk powder, fruit juice, soybean milk, vegetable-fruit juice, healthfood, animal feed, an agricultural product, a livestock product, anaquatic product, and a functional ingredient. In a preferred embodimentof this invention, the edible material used as the medium is milk. Inanother preferred embodiment of this invention, the edible material usedas the medium is orange juice. In yet another preferred embodiment ofthis invention, the edible material used as the medium is soybean milk.

According to this invention, the suitable medium comprises a carbonsource selected from the group consisting of: lactose, fructose,maltose, glucose, molasses, galactose, xylose, xylitol, inulin,sorbitol, trehalose, sucrose, and mixtures thereof. Preferably, thecarbon source is selected from the group consisting of: maltose,glucose, molasses, xylose, inulin, sucrose, and mixtures thereof. Morepreferably, the carbon source is selected from the group consisting of:inulin, sucrose, and mixtures thereof. In the preferred embodiments ofthis invention, the carbon source is sucrose.

Preferably, before cultivating, the suitable medium has a pH valueranging from 3 to 7. In a preferred embodiment of this invention, beforecultivating, the suitable medium has a pH value of 5.

Preferably, the cultivating step for Pediococcus acidilactici 05B0111 isconducted at a temperature ranging from 25° C. to 37° C. In a preferredembodiment of this invention, Pediococcus acidilactici 05B0111 iscultivated at 30° C.

Isolation and purification of exopolysaccharides, and determination ofexopolysaccharide concentration can be performed by virtue of techniquesknown to a skilled artisan. For example, exopolysaccharides can beisolated and purified using trichloroacetic acid or ethanol.Exopolysaccharide concentration can be determined using phenol-sulfuricmethod (P. Ruas-Madiedo and C. G. de los Reyes-Gavilán (2005), supra).

Exopolysaccharides isolated and purified from culture. of Pediococcusacidilactici 05B0111 have been used to culture macrophages and have beenhence shown to have immunomodulating activity. Moreover, based on otherknown probiotic properties of exopolysaccharides, the culture ofPediococcus acidilactici 05B0111, which contains exopolysaccharides, isexpected to be applicable to production of drugs for immune modulation,lowering cholesterol, and antitumor.

Accordingly, this invention provides a pharmaceutical compositioncomprising the isolated Pediococcus acidilactici 05B0111. Preferably,the pharmaceutical composition further comprises the exopolysaccharideobtained from the aforementioned method of producing anexopolysaccharide.

The pharmaceutical composition may be orally administrable and can beformulated into a dosage form of e.g., solution, suspension, emulsion,powder, a tablet, a pill, syrup, lozenge, troche, chewing gum, acapsule, slurry, and an analogue thereof. Preferably, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier that is not able to induce an allergic reaction or anundesired effect in a subject when being administered to the same.According to this invention, the pharmaceutically acceptable carrier mayinclude one or more of the following reagents: a solvent, emulsifier, asuspending agent, decomposer, a binding agent, an excipient, astabilizing agent, a chelating agent, diluent, a gelling agent,preservative, lubricant, and an analogue thereof.

Besides the oral dosage form, the pharmaceutical composition containingthe isolated Pediococcus acidilactici 05B0111 can also be formulatedinto a suitable dosage form for topical administration (e.g.,percutaneous administration or intranasal administration). Examples ofthe suitable dosage form for topical administration include, but are notlimited to cream, lotion, ointment, nasal spray formulation, aerosol,and dry powder inhaler.

Additionally, this invention provides a food product that comprises anedible material and the isolated Pediococcus acidilactici 05B0111.Preferably, the food product further comprises the exopolysaccharideobtained from the aforementioned method of producing anexopolysaccharide. Examples of the edible material contained in the foodproduct include, but are not limited to, milk powder, a beverage,confectionery, a candy, a fermented food, animal feeds, a health food, adietary supplement, jelly, infant formula, salad dressing, mayonnaise,spread, cream, a sauce, a pudding, ice-cream, a bakery product, ketchup,and mustard.

The culture medium of Pediococcus acidilactici 05B0111, which containsexopolysaccharides, is expected to be applicable to production of healthfood and non-prescribed drugs for immune modulation, loweringcholesterol, and antitumor. Regarding the food product of thisinvention, the edible material thereof can be used as a medium forcultivation of the isolated Pediococcus acidilactici 05B0111.

This invention will be further described by way of the followingexamples. However, it should be understood that the following examplesare solely intended for the purpose of illustration and should not beconstrued as limiting the invention in practice.

EXAMPLES Experimental Materials: A. MRS Broth:

MRS broth to be used in the following examples is commercially availableLactobacilli MRS broth (DIFCO, Cat. No. 0881) that contains 2% glucoseas a carbon source.

B. MRS-Carbohydrate Broth:

MRS-carbohydrate broth to be used in the following examples isformulated mainly according to the recipe of MRS broth, except that acarbohydrate other than glucose is used as a carbon source, and thatcarbohydrate concentration of the MRS-carbohydrate broth is 10%. Forinstance, the MRS-sucrose broth contains 10% sucrose instead of 2%glucose.

C. Beverage Containing 10% (v/v) Sucrose:

Beverages to be used in the following examples are orange juice(pH=3.89) (purchased from Chia Meei Food Indl. Corp.), whole milk(purchased from Kuang Chuan Dairy Co., Ltd.), and soybean milk withoutsugar (purchased from I-Mei Foods Co., Ltd.).

First, a proper amount of sucrose was dissolved in ddH₂O so as to make asucrose stock solution having a concentration of 50% (v/v), followed bysterilization at 121° C. for 15 minutes. The 50% sucrose stock solutionwas then cooled down for further use. The orange juice was placed in awater bath of 90° C. for 1 minute for sterilization. Furthermore, eachof the whole milk and the soybean milk without sugar was placed in awater bath of 100° C. for 30 minutes for sterilization. The threebeverages were cooled down to room temperature. A portion of the orangejuice (pH=3.89) was obtained, and pH thereof was adjusted to 5.0 usingNaOH.

A proper amount of the 50% sucrose stock solution was added to each ofthe sterile orange juice (pH=3.89 and pH=5), and to each of the sterilewhole milk and the sterile soybean milk without sugar so that orangejuice (pH=3.89 and pH=5) containing 10% sucrose, milk containing 10%sucrose, and soybean milk containing 10% sucrose were prepared.

General Experimental Procedures: A. Isolation and Purification ofExopolysaccharides:

A test sample having a predetermined volume and 20% trichloroacetic acidhaving an equivalent volume relative to the test sample were evenlymixed at 200 rpm for 2 hours using a suspension mixer, followed bycentrifugation at 3000 rpm for 20 minutes. The resultant supernatant wasacquired and was mixed with 95% alcohol (4° C.) having a 2-fold volumerelative to the supernatant. The resultant mixture was left at 4° C.overnight and was subsequently centrifuged at 3000 rpm for 20 minutes,followed by removing supernatant. Precipitate was dissolved in ddH₂Ohaving an equivalent volume relative to the precipitate, followed byaddition of 95% alcohol (4° C.) having a 2-fold volume relative to theresultant solution and overnight storage at Centrifugation at 3000 rpmfor 20 minutes was performed, and supernatant was removed. Precipitatewas freeze-dried so as to obtain gelatinous or membranousexopolysaccharides (EPS) for further use.

B. Determination of EPS Concentration:

EPS obtained according to the procedures as described in the precedingsection, entitled “A. Isolation and purification of exopolysaccharides”,were dissolved in ddH₂O having a volume equivalent to the predeterminedvolume of the test sample as mentioned in the preceding section,entitled “A. Isolation and purification of exopolysaccharides”.Therefore, an EPS solution was formed. 1 mL of the EPS solution wastaken out, followed by sequentially adding 0.5 mL of 5% phenol solutionand 2.5 mL of concentrated sulfuric acid, and mixing evenly. Theresultant mixture was reacted at room temperature for 10 minutes and wassubsequently placed in a water bath of 30° C. for 15 minutes. Finally,an ELISA reader (SpectraMax M2, Molecular Devices) was used to measureabsorbance (OD₄₉₀) of the resultant mixture at a wavelength of 490 nm.OD₄₉₉ of the resultant mixture was converted to a concentration (mg/L)according to a correlation curve that was generated beforehand byplotting concentrations (0 mg/L, 20 mg/L, 40 mg/L, 80 mg/L, and 100mg/L) of EPS standards and respective OD₄₉₀.

Example 1 Screening of Lactic Acid Bacteria Isolates HavingEPS-Producing Ability A. Source and Isolation of Test Strains:

Lactic acid bacteria strains were isolated from several fermented foodproducts (such as Chinese pickle, kimchi, Chinese cheese, preservedplum, other preserved food products, etc.) purchased from a traditionalmarket. A proper amount of the selected fermented food product was addedin MRS broth, followed by cultivation at 37° C. for 1 to 3 days.Subsequently, the resultant bacteria culture was subjected to 10-foldserial dilution so that 10¹˜10⁷-fold diluted bacteria culture solutionswere formed. 0.2 mL of each of the 10²-, 10³-, 10⁴-, 10⁵-, 10⁶-, and10⁷-fold diluted bacteria culture solutions was obtained and was evenlyapplied onto a MRS agar plate, followed by cultivation at 37° C. for 72hours. Afterward, based on colonial morphology and the results ofmicroscopic examination, the applicants selected 121 lactic acidbacteria isolates and purified the same several times. Each of thepurified lactic acid bacteria isolates was mixed with 20% glycerol. Theresultant mixture was placed in a frozen vial and was stored at −80° C.for further use.

B. Preparation of Inoculums of Lactic Acid Bacteria Isolates:

Each of the lactic acid bacteria isolates as obtained from the precedingsection, entitled “A. Source and isolation of test strain”, wasinoculated in 5 mL of MRS broth so that the MRS broth had the finalinoculum concentration of 1% (v/v), followed by cultivation at 37° C.for 18 hours. The aforementioned cultivation step was repeated twice soas to activate the lactic acid bacteria isolates. The resultant cultureserved as inoculums of the lactic acid bacteria isolate in the followingexamples.

C. Preliminary Screening of Lactic Acid Bacteria Isolates HavingEPS-Producing Ability:

The inoculums of each of the 121 lactic acid bacteria isolates, whichwere obtained from the preceding section entitled “B. Preparation ofinoculums of lactic acid bacteria isolates”, were inoculated in EPSselection medium (ESM) so that the ESM had a final inoculumconcentration of 1% (v/v), followed by cultivation at 37° C. for 72hours. A proper amount of the resultant culture was taken out todetermine the viscosity thereof using a viscometer (model DV-I+,BROOKFIELD, USA).

In addition, a proper amount of the inoculums of each of the 121 lacticacid bacteria isolates, which were obtained from the preceding sectionentitled “B. Preparation of inoculums of lactic acid bacteria isolates”,were applied onto a MRS-sucrose agar plate by virtue of thefour-quadrant streak method, followed by cultivation at 37° C. for 5days. Physical appearance of the colonies was observed by human vision,and the colonies were picked with tips of toothpicks so as to seewhether the colonies could produce ropy strands.

When a lactic acid bacteria isolate meets at least one of the twofollowing requirements, the same is considered as a strain capable ofproducing EPS:

-   -   (1) Viscosity is over 150 cp; and    -   (2) Slime is observed around colonies, or colonies produce ropy        strands when the same are picked with tips of toothpicks.

According to the aforementioned requirements and consideration forfactors (such as a source of strains and suitability of strains todevelop a food product), the applicants preliminarily screened 26isolates from the 121 lactic acid bacteria isolates and performed 16SrDNA sequence analysis for the 26 isolates so as to determine thespecies thereof.

D. 16S rDNA Sequence Analysis:

The inoculums of each of the 26 lactic acid bacteria isolates, whichwere obtained from the preceding section entitled “B. Preparation ofinoculums of lactic acid bacteria isolates”, were added onto a MRS agarplate and were evenly spread using a L-shaped glass rod, followed bycultivation at 37° C. for 24 hours. Afterward, a small amount of freshstrains were scraped from the MRS agar plate and transferred to 1.5 mLmicrocentrifuge tube. Genomic DNA of the bacterial strain was extractedusing Blood & Tissue Genomic DNA Extraction Miniprep System (Viogene,U.S.A.). Extracted genomic DNA was dissolved in a proper amount ofddH₂O. Therefore, 26 samples, each of which includes genomic DNA of arespective one of the 26 lactic acid bacteria isolates, were formed.

The genomic DNA thus obtained was used as a template in a 16S rDNA PCR(polymerase chain reaction) experiment using GeneAmp® PCR system 9700(Applied Biosystem) and MicroSeq® Full Gene 16S rDNA BacterialIdentification PCR Kit (Applied Biosystem). After the PCR experiment wascompleted, 1% agarose gel electrophoresis was performed to detect a PCRamplification product of about 500 bps. By virtue of PCR Clean up-MSystem (Viogene), the detected PCR amplification product was recycledand purified from the gel.

The purified PCR amplification product was subjected to a cyclesequencing experiment using GeneAmp® PCR system 9700 and MicroSeq® FullGene 16S rDNA Bacterial Identification Sequencing Kit (AppliedBiosystem). The cycle sequencing product was subsequently purified usingPerforma™ DTR Gel Filtration Systems Gel Filtration Cartridges (EdgeBioSystems). The thus purified cycle sequencing product was sequencedusing 3730 DNA Analyzer (Applied Biosystems), and the resultant 16S rDNAsequence was compared to the database on NCBI website using MicroSeq®IDanalysis software (v. 1.40, Applied Biosystems). The result ofcomparison is shown in Table 1.

TABLE 1 Comparison of 16S rDNA sequence of lactic acid bacteria isolatesNumber of lactic acid Homology bacteria isolate Species* (%) 05B0001 (a)Lactobacillus kimchii 99 (b) Lactobacillus paralimentarius 05B0002Pediococcus sp. 99 05B0003 Pediococcus pentosaceus 99 05B0025Lactobacillus fermentum 99 05B0100 Lactobacillus plantarum 99 05B0108Pediococcus pentosaceus 99 05B0111 Pediococcus acidilactici 99 05B0114(a) Streptococcus macedonicus 99 (b) Streptococcus waius 05B0115Weissella paramesenteroides 99 05B0116 Enterococcus faecalis 99 05B0117Lactobacillus gasseri 99 05B0164 Pediococcus acidilactici 99 05B0165Pediococcus pentosaceus 99 05B0166 Lactobacillus pontis 98 05B0167Pediococcus acidilactici 98 05B0168 Pediococcus acidilactici 99 05B0169Lactobacillus plantarum 99 05B0G5-2 Lactobacillus brevis 99 05B013-2Lactobacillus plantarum 99 05B017-1 Lactobacillus fermentum 99 05B017-2Lactobacillus delbrueckii subsp. 99 bulgaricus 05B018-1 Lactobacillusfermentum 99 05B029-2 Lactobacillus plantarum 99 05B033-1 Weissellaparamesenteroides 99 05B034-2 Pediococcus acidilactici 98 05B046-2 (a)Lactobacillus amylovorus 99 (b) Lactobacillus kitasatonis (c)Lactobacillus sobrius (d) Lactobacillus gallinarum *The column “species”indicates a species name (i.e., a scientific name) of a type strainhaving 16S rDNA sequence that is most homologous to 16S rDNA sequence ofthe respective isolate of this invnetion. If more than one species nameappear in the aforementioned column, it indicates that the isolate ofthis invention has 16S rDNA sequence most homologous to 16S rDNAsequence of more than one type strain.

Referring to Table 1, the results of comparison between 16S rDNAsequences show: 13 isolates belong to the genus Lactobacillus, 9isolates belong to the genus Pediococcus, 2 isolates belong to the genusWeissella, 1 isolate belongs to the genus Streptococcus, and 1 isolatebelongs to the genus Enterococcus. Particularly, the applicants havenoticed that among the 9 isolates belonging to the genus Pediococcus, 5isolates (numbers thereof are 05B0111, 05B0164, 05B0167, 05B0168, and05B034-2) are Pediococcus acidilactici. None of the previous literaturesand prior art have disclosed that Pediococcus acidilactici hasEPS-producing ability.

In order to massively produce EPS, the applicants decided to furtherscreen strains having great EPS-producing ability. Thus, the 26 lacticacid bacteria isolates were subjected to the following experiments.

E. Screening of Lactic Acid Bacteria Isolates Having Great EPS-ProducingAbility:

The inoculums of each of the 26 lactic acid bacteria isolates asobtained from the preceding section, entitled “C. Preliminary screeningof lactic acid bacteria isolates having EPS-producing ability”, wereinoculated in 15 mL of MRS-sucrose broth so that the MRS-sucrose brothhad a final inoculum concentration of 1% (v/v), followed by cultivationat 37° C. for 72 hours. Isolation and purification of EPS in theresultant culture, and determination of EPS concentration of theresultant culture were performed according to the procedures asdescribed in the sections, entitled “A. Isolation and purification ofexopolysaccharides” and “B. Determination of EPS concentration”, of theGeneral Experimental Procedures.

The results show that the cultures of the 5 Pediococcus acidilacticiisolates (numbers thereof 05B0111, 05B0164, 05B0167, 05B0168, and05B034-2) as preliminarily identified in the preceding section, entitled“D. 16S rDNA sequence analysis”, respectively have the following EPSconcentrations: 845 mg/L, 11.34 mg/L, 29.51 mg/L, 17.99 mg/L, and 17.74mg/L. Furthermore, among the remaining 21 lactic acid bacteria isolates,cultures of 4 isolates have EPS concentrations ranging between 60 mg/Land 110 mg/L, and cultures of the rest of the bacteria isolates have EPSconcentrations that are ranging between 50 mg/L and 2.89 mg/L. Based onthe results of this experiment, the applicants believe that Pediococcusacidilactici 05B0111 has the greatest potential for producing EPS.Pediococcus acidilactici 05B0111 was further subjected to assessment ofphylogenetic novelty.

Example 2 Assessment of Phylogenetic Novelty of Pediococcus Acidilactici05B0111

In order to confirm that Pediococcus acidilactici 05B0111 as screened inthe above Example 1 has phylogeny different from that of the publishedstrains belonging to the genus Pediococcus, Pediococcus acidilactici05B0111 was subjected to the following preliminary tests, 16S rDNAsequence analysis, physiological and biochemical tests, and DNA-DNAhybridization analysis.

Experimental Materials and Procedures: A. Preliminary Tests:

The preliminary tests for Pediococcus acidilactici 05B0111 include Gramstain, morphological observation, motility, activity of catalase andoxidase, growth condition under aerobic and anaerobic environments, andability to produce endospore.

The results show: Pediococcus acidilactici 05B0111 is Gram-positive,does not have motility, does not have activity of catalase and oxidase,is able to grow under aerobic and anaerobic environments, and is notable to produce endospore.

B. 16S rDNA Sequence Analysis:

16S rDNA of Pediococcus acidilactici 05B0111 was sequenced according tothe procedures as described in the section, entitled “D. 16S rDNAsequence analysis”, of the above Example 1. The full-length sequence of16S rDNA of Pediococcus acidilactici 05B0111 is shown in FIG. 1, and wascompared to the database on NCBI website using MicroSeq®ID analysissoftware. The comparison shows: the sequence similarity between thefull-length sequence (SEQ ID NO:1) of 16S rDNA of Pediococcusacidilactici 05B0111 and 16S rDNA sequence of Pediococcus (Genbankaccession number: AJ249535), the sequence similarity between thefull-length sequence of 16S rDNA of Pediococcus acidilactici 05B0111 and16S rDNA sequence of Pediococcus stilesii (Genbank accession number:AJ973157), and the sequence similarity between the full-length sequenceof 16S rDNA of Pediococcus acidilactici 05B0111 and 16S rDNA sequence ofPediococcus claussenii (Genbank accession number: AF404716) are all over97%.

C. Physiological and Biochemical Tests:

Temperature tolerance, pH tolerance, NaCl tolerance, and carbohydratefermentation profile of Pediococcus acidilactici 05B0111 were tested.

The procedures of the tests for temperature tolerance, pH tolerance, andNaCl tolerance were slightly modified according to the procedures asdescribed by Charles M. A. P. Franz et al. (Charles M. A. P. Franz etal. (2006), International Journal of Systematic and EvolutionaryMicrobiology, 56:329-333). In a nutshell, a proper amount of theinoculums of Pediococcus acidilactici 05B0111, which were prepared inthe section entitled “B. Preparation of inoculums of lactic acidbacteria isolates” of the above Example 1, were added onto a MRS agarplate and were evenly spread using a L-shaped glass rod, followed bycultivation at 37° C. for 5 days. Afterward, growth condition ofPediococcus acidilactici 05B0111 on the MRS agar plate was observed.

Carbohydrate fermentation profile was tested using API 50 CHLidentification system (bioMérieux) and the manual guide thereof. Thefollowing carbohydrates were tested: arabinose, galactose, lactose,maltose, ribose, xylose, glycerol, potassium gluconate, potassium 2-ketogluconate, and trehalose.

In addition, due to the results of the section, entitled “B. 16S rDNAsequence analysis”, of the Example 2, the applicants used threePediococcus type strains purchased from BCRC of FIRDI as comparativestrains to contrast with Pediococcus acidilactici 05B0111. The threePediococcus type strains are as follows:

-   -   (1) Pediococcus acidilactici BCRC 17599^(T) (corresponding to        DSM 20284^(T)) isolated from barley,    -   (2) Pediococcus claussenii BCRC 17600^(T) (corresponding to DEM        14800^(T), ATCC BAA-344^(T), and KCTC 3811^(T)) isolated from        spoiled beer, and    -   (3) Pediococcus stilesii BCRC 17601^(T) (corresponding to DSM        18001^(T), CCUG 51290^(T), and LMG 23082^(T)) isolated from        white maize grains.

Temperature tolerance, pH tolerance, NaCl tolerance, and carbohydratefermentation profile regarding Pediococcus acidilactici 05B0111 and thethree Pediococcus type strains are shown in Table 2.

TABLE 2 Physiological and biochemical tests for Pediococcus acidilactici05B0111 and three Pediococcus type strains Pediococcus PediococcusPediococcus acidilactici claussenii Pediococcus acidilactici BCRC BCRCstilesii BCRC Test 05B0111 17599^(T) 17600^(T) 17601^(T) pH pH4.2 + + + + tolerance* pH 7.0 + + + + pH 8.0 + + + + pH 9.0 + − − +Temperature 35° C. + + + + tolerance* 40° C. + + + + 45° C. + − − + 50°C. + − − − NaCl  4% NaCl + + + + Tolerance*  5% NaCl + + + +  6%NaCl + + − +  8% NaCl + + − + 10% NaCl − + − − Carbohydrate Arabinose− + − − fermentation Galactose + + − + profile** Lactose − − − − Maltose− − + + Ribose + + + + Xylose − + − − Glycerol − − − + Potassium − − − +gluconate Potassium − − − + 2-keto gluconate Trehalose + + + −*Regarding results of the tests for pH tolerance, temperature tolerance,and NaCl tolerance, “+” indicates that the test strain is able to growon the MRS agar plate with the respective condition, and “−” indicatesthat the test strain is not able to grow on the MRS agar plate with therespective condition. **Regarding results of the tests for carbohydratefermentation profile, “+” indicates that the test strain is capable ofusing the respective carbohydrate to perform fermentation and produceacid, and “−” indicates that the test strain is not capable of using therespective carbohydrate to perform fermentation.

Based on the results shown in Table 2, the applicants believe that thephysiological and biochemical characteristics of Pediococcusacidilactici 05B0111 are generally conformable to those of strainsbelonging to Pediococcus, and are similar to those of Pediococcusacidilactici BCRC 17599^(T) , Pediococcus claussenii BCRC 17600^(T), andPediococcus stilesii BCRC 17601^(T).

D. DNA-DNA Hybridization Analysis:

In order to further confirm the species of Pediococcus acidilactici05B0111, the applicants prepared genomic DNA samples of Pediococcusacidilactici 05B0111, Pediococcus acidilactici BCRC 17599^(T) ,Pediococcus claussenii BCRC 17600^(T), and Pediococcus stilesii BCRC17601^(T) according to the procedures as described in the section,entitled “D. 16S rDNA sequence analysis”, of the above Example 1. Theapplicants conducted DNA-DNA hybridization according to the proceduresas described by Takayuki Ezaki et al. (Takayuki Ezaki et al. (1989),International Journal of Systematic Bacteriology, 39:224-229). First,each of the genomic DNA samples was subjected to heat-denaturing,followed by mixing with phosphate buffered saline (PBS) containing 0.1 MMgCl₂ so as to make a DNA solution having a concentration of 20 μg/mL.The DNA solution of Pediococcus acidilactici 05B0111 was designated asthe 05B0111 group, and the DNA solutions of Pediococcus acidilacticiBCRC 17599^(T) , Pediococcus claussenii BCRC 17600^(T), and Pediococcusstilesii BCRC 17601^(T) were designated as the type strain groups.Furthermore, a DNA solution (Merck, Cat. No. 2618) of calf thymus servedas the negative control group. 100 μL of the DNA solution of each groupwas added into a respective well of a 96-well plate, followed bycultivation at 37° C. for 1 hour. Each of the DNA solutions was removedfrom the respective well. The wells were rinsed using PBS containing 0.1M MgCl₂. The 96-well plate was dried at 60° C. overnight.

In addition, 5 μL of photobiotin and 5 μL of the heat-denatured genomicDNA sample (1 μg/μL, in distilled water) of Pediococcus acidilactici05B0111 were added into a microcentrifuge tube and evenly mixed,followed by illumination for 15 minutes by a sunlamp (500 W). Freephotobiotin was removed by virtue of 2-butanol extraction. Consequently,biotinylated DNA was formed and was kept for further use.

The 96-well plate that was dried overnight was obtained. 200 μL of aprehybridization solution (containing 2×SSC, 5× Denhardt solution,denatured salmon sperm DNA (200 μg/mL), and 50% formamide) was addedinto each of the wells coated with the respective DNA solutions,followed by cultivation at 37° C. for 1 hour. The liquid in each of thewells was removed. Subsequently, 100 μL of a hybridization solution(containing 2×SSC, 5× Denhardt solution, 3% dextran sulfate, 50%formamide, denatured salmon DNA (50 μg/mL), and 50 ng of thebiotinylated DNA) was added into each of the wells, followed byhybridization reaction at 42° C. for 1 hour. The liquid in each of thewells was removed. Each of the wells was washed four times using 300 μLof 2×SSC buffer solution, followed by adding 100 μL ofstreptavidin-beta-D-galactosidase solution (10 ng/mL, in PBS containing0.5% bovine serum albumin and 0.1% Triton X-100). The resultant mixtureswere allowed to react at 37° C. for 30 minutes.

Afterward, the liquid in each of the wells was removed, and each of thewells was rinsed twice with PBS containing 0.1% Triton X-100. 100 μL of4-methylumbelliferyl-beta-D-galactopyranoside (3×10⁻⁴ M, in PBS) wasadded into each of the wells. The resultant mixtures were allowed toreact at 37° C. for 10 minutes. Fluorescence intensity of the resultantmixture in each of the wells was measured at 360 nm (excitationwavelength) and 450 nm (emission wavelength) using Fluoroskan IImicroplate fluorometer (Labsystems).

DNA relatedness between Pediococcus acidilactici 05B0111 and Pediococcusacidilactici BCRC 17599^(T), DNA relatedness between Pediococcusacidilactici 05B0111 and Pediococcus claussenii BCRC 17600^(T), and DNArelatedness between Pediococcus acidilactici 05B0111 and Pediococcusstilesii BCRC 17601^(T) were calculated by substituting the fluorescenceintensity as measured in this section into the following formula:

DNA relatedness (%=[(A−C)/(B−C)]×100

where: A=fluorescence intensity of the type strain group

-   -   B=fluorescence intensity of the 05B0111 group    -   C=fluorescence intensity of the negative control group

The results show that DNA relatedness between the genomic DNAs ofPediococcus acidilactici 05B0111 and Pediococcus acidilactici BCRC17599^(T), DNA relatedness between the genomic DNAs of Pediococcusacidilactici 05B0111 and Pediococcus claussenii BCRC 17600^(T), and DNArelatedness between the genomic DNAs of Pediococcus acidilactici 05B0111and Pediococcus stilesii BCRC 17601^(T) are 75.5%, 21.1%, and 4.9%,respectively. Based on the results, the applicants confirmed that theisolate 05B0111 is Pediococcus acidilactici.

Based on the results of the aforementioned tests and analysis, Bergey'sManual of Determinative Bacteriology (Holt et al. (1994), Bergey'sManual of Determinative Bacteriology, 9^(th) edition, Williams &Wilkins), and the microbiology literatures (e.g., Franz et al. (2006),International Journal of Systematic and Evolutionary Microbiology,56:329-333), the applicants deem that Pediococcus acidilactici 05B0111of this invention is a novel Pediococcus acidilactici isolate.

Pediococcus acidilactici 05B0111 of this invention was deposited in BCRCof FIRDI (331 Shih-Pin Road, Hsinchu 300, Taiwan) under the accessionnumber BCRC 910420 on Feb. 10, 2009. Pediococcus acidilactici 05B0111was also deposited in DSMZ under the accession number DSM 22345 on Mar.5, 2009 so as to meet the requirement of the Budapest Treaty.

Example 3 Acid Tolerance Test and Bile Salt Tolerance Test forPediococcus Acidilactici 05B0111

Pediococcus acidilactici 05B0111 of this invention was subjected to thefollowing experiments so as to investigate whether the same is able tosurvive under the strict condition of the digestive tract after beingingested.

Experimental Procedures: A. Acid Tolerance Test:

A proper amount of the inoculums of Pediococcus acidilactici 05B0111,which were obtained from the section entitled “B. Preparation ofinoculums of lactic acid bacteria isolates” of the above Example 1, wereinoculated into 10 mL of MRS broth, followed by cultivation at 37° C.for 24 hours and centrifugation at 3000 rpm for 10 minutes. Thesupernatant was then removed, and 1 mL of PBS was added to sufficientlysuspend strains. Thus, a suspension was made 0.5 mL of the suspensionwas inoculated into each of two PBS solutions (pH=2 and pH=7.2) having avolume of 10 mL, and was sufficiently mixed with the same. The resultantmixture was placed at 37° C. for 2 hours for cultivation. 1 mL of theresultant culture was obtained and was subjected to 10-fold serialdilution using PBS, followed by a spread plate procedure to count theviable bacteria.

B. Bile Salt Tolerance Test:

The inoculums of Pediococcus acidilactici 05B0111, which were obtainedfrom the section entitled “B. Preparation of inoculums of lactic acidbacteria isolates” of the above Example 1, were inoculated into each of10 mL of MRS broth and 10 mL of MRS broth containing 0.3% (v/v) oxgall(DIFCO) so that the MRS broth without oxgall and the MRS brothcontaining 0. 3% oxgall had final inoculum concentrations of 2% (v/v),followed by mixing sufficiently. The resultant mixture was placed at 37°C. for 24 hours for cultivation. 1 mL of the resultant culture wassubjected to 10-fold serial dilution using PBS, followed by a spreadplate procedure to count the viable bacteria.

Results:

When the PBS solutions having pH 2 and pH 7.2 were used to cultivatePediococcus acidilactici 05B0111 of this invention for 2 hours, theviable bacteria counts are respectively 9.05 log CFU/mL and 9.40 logCFU/mL. In addition, when the MRS broth without oxgall and the MRS brothcontaining 0.3% (v/v) oxgall were used to cultivate Pediococcusacidilactici 05B0111 of this invention for 24 hours, the viable bacteriacounts are respectively 9.07 log CFU/mL and 8.40 log CFU/mL. The resultsprove that Pediococcus acidilactici 05B0111 of this invention is able totolerate acid and bile salt, and is hence capable of tolerating theenvironment of the human digestive tract after being ingested.

Example 4 Effects of Different Cultivation Conditions on EPS-ProducingAbility of Pediococcus Acidilactici 05B0111 A. Effects of Carbohydrateson EPS-Producing Ability of Pediococcus Acidilactici 05B0111:

12 kinds of carbohydrates (i.e., lactose, fructose, maltose, glucose,molasses, galactose, xylose, xylitol, inulin, sorbitol, trehalose, andsucrose) were used as carbon sources so as to investigate effectsthereof on EPS-producing ability of Pediococcus acidilactici 05B0111.

The inoculums of Pediococcus acidilactici 05B0111, which were obtainedin the section entitled “B. Preparation of inoculums of lactic acidbacteria isolates” of the above Example 1, were inoculated into each of12 kinds of MRS-carbohydrate broth (each has 15 mL) so that each of the12 kinds of MRS-carbohydrate broth had a final inoculum concentration of1% (v/v), followed by cultivation at 37t for 72 hours. Isolation andpurification of EPS in the resultant culture, and determination of EPSconcentration of the resultant culture were conducted according to theprocedures as described in the sections, entitled “A. Isolation andpurification of exopolysaccharides” and “B. Determination of EPSconcentration”, of the above General Experimental Procedures. Theresults are shown in FIG. 2.

Referring to FIG. 2, EPS-producing ability of Pediococcus acidilactici05B0111 varies with the different carbon sources. Particularly, comparedto other carbohydrates, EPS concentration is the highest (about 850mg/L) when sucrose serves as a carbon source. The results indicate thatwhen Pediococcus acidilactici 05B0111 of this invention utilizes sucroseas a carbon source for growth and as an energy source, the same is ableto produce a large number of EPS.

B. Effects of Temperatures on EPS-Producing Ability of PediococcusAcidilactici 05B0111:

Effects of different cultivation temperatures on EPS-producing abilityof Pediococcus acidilactici 05B0111 were examined when sucrose served asa carbon source.

The inoculums of Pediococcus acidilactici 05B0111, which were obtainedin the section entitled “B. Preparation of inoculums of lactic acidbacteria isolates” of the above Example 1, were inoculated into each oftwo portions (each had 5 L) of orange juice containing 10% sucrose sothat each of the two portions of the orange juice had a final inoculumconcentration of 4% (v/v), followed by placing the two portions of theorange juice respectively at 30° C. and 37° C. for cultivation. At 8 hrsand 72 hrs, a part of the resultant culture was obtained; and isolationand purification of EPS in the resultant culture, and determination ofEPS concentration of the resultant culture were conducted according tothe procedures as described in the sections, entitled “A. Isolation andpurification of exopolysaccharides” and “B. Determination of EPSconcentration”, of the above General Experimental Procedures. Theresults are shown in FIG. 3.

Referring to FIG. 3, at 8 hrs and 72 hrs, EPS concentration of theculture cultivated at 30° C. is higher than that of the culturecultivated at 37° C. The results show that when Pediococcus acidilactici05B0111 of this invention uses sucrose as a carbon source and iscultivated at 30° C., the same has better EPS-producing ability.

C. Effects of Initial pH Values on EPS-Producing Ability of PediococcusAcidilactici 05B0111:

Effects of different initial pH values on EPS-producing ability ofPediococcus acidilactici 05B0111 were investigated when sucrose servedas a carbon source and cultivation was conducted at 30° C.

The inoculums of Pediococcus acidilactici 05B0111, which were obtainedin the section entitled “B. Preparation of inoculums of lactic acidbacteria isolates” of the above Example 1, were inoculated into 80 mL ofeach of the two portions of the orange juice (both contained 10% sucroseand respectively had initial pH values of 3.89 and 5.0) so that each ofthe two portions of the orange juice had a final inoculum concentrationof 4% (v/v), followed by placing at 30° C. for 72 hours for cultivation.Isolation and purification of EPS in the resultant culture, anddetermination of EPS concentration of the resultant culture wereperformed according to the procedures as described in the sections,entitled “A. Isolation and purification of exopolysaccharides” and “B.Determination of EPS concentration”, of the above General ExperimentalProcedures.

As a result, EPS concentration of the culture cultivated using themedium with the initial pH value of 3.89 is 677.33 mg/L, and EPSconcentration of the culture cultivated using the medium with theinitial pH value of 5.0 is 1154 mg/L. The results prove that whenPediococcus acidilactici 05B0111 of this invention utilizes sucrose as acarbon source and is cultivated at 30° C. using a medium with an initialpH value of 5.0, the same has better EPS-producing ability.

Example 5 Evaluation of EPS-Producing Ability of PediococcusAcidilactici 05B0111 in Different Beverages

In order to examine how EPS concentration of beverages containing 10%(v/v) sucrose is affected by addition of Pediococcus acidilactici05B0111 of this invention, first, isolation and purification of EPS, anddetermination of EPS concentration were performed according to theprocedures as described in the sections, entitled “A. Isolation andpurification of exopolysaccharides” and “B. Determination of EPSconcentration”, of the above General Experimental Procedures for aproper amount of each of the following beverages containing 10% (v/v)sucrose: orange juice, milk, and soybean milk.

Afterward, the inoculums of Pediococcus acidilactici 05B0111, which wereobtained in the section entitled “B. Preparation of inoculums of lacticacid bacteria isolates” of the above Example 1, were inoculated intoeach of the orange juice (5 L) containing 10% sucrose, the milk (30 mL)containing 10% sucrose, and the soybean milk (50 mL) containing 10%sucrose so that the orange juice, the milk, and the soybean milkrespectively had final inoculum concentrations (v/v) of 4%, 2%, and 2% .The orange juice with the inoculums was placed at 30° C. for 72 hours ofcultivation, and the milk with the inoculums and the soybean milk withthe inoculums were placed at 37° C. for 72 hours for cultivation.Isolation and purification of EPS in the resultant culture, anddetermination of EPS concentration of the resultant culture wereconducted according to the procedures as described in the sections,entitled “A. Isolation and purification of exopolysaccharides” and “B.Determination of EPS concentration”, of the above General ExperimentalProcedures.

Before inoculation with Pediococcus acidilactici 05B0111 of thisinvention, EPS concentrations of the orange juice containing 10%sucrose, the milk containing 10% sucrose, and the soybean milkcontaining 10% sucrose are respectively 452 mg/L, 10 mg/L, and 157 mg/L.After the inoculation with Pediococcus acidilactici 05B0111 of thisinvention and cultivation for 72 hours, EPS concentrations of thecultures cultivated using the orange juice, the milk, and the soybeanmilk are respectively 677 mg/L, 655 mg/L, and 1128 mg/L. The resultsindicate that Pediococcus acidilactici 05B0111 of this invention hasEPS-producing ability in different beverages and is able to be widelyused in the food industry.

Example 6 Evaluation for Ability of EPS Produced by PediococcusAcidilactici 05B0111 to Stimulate Proinflammatory Cytokine Secretion andNitric Oxide Secretion of Mouse Macrophage

Mouse macrophages were cultivated with EPS produced by Pediococcusacidilactici 05B0111 of this invention. In this example, Interleukin-6(IL-6), interleukin-10 (IL-10), monocyte chemoattractant protein-1(MCP-1), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), andinterleukin-12P70 (IL-12p70) were regarded as indices of expressionlevel of proinflammatory cytokines, and nitrite served as an index oflevel of nitric oxide (NO).

Experimental Materials and Procedures: A. Source and Cultivation ofMacrophage:

13.4 g of Dulbecco's modified Eagle's medium (DMEM) powder (HyClone) and1.5 g of sodium bicarbonate (NaHCO₃) were respectively added into 600 mLof ddH₂O and 300 mL of ddH₂O. After sufficient dissolution, the tworesultant mixtures were evenly blended together, followed by adjustingpH to 7.2˜7.4 by virtue of 1N HCl. Therefore, DMEM medium was prepared.10% heat inactivated fetal bovine serum (FBS) and 2% glutamine wereadded into DMEM, followed by filtration using a membrane filter(Millipore) having a pore size of 0.22 μm and storage at 4° C.

In this example, mouse BALB/c macrophage RAW 264.7 (purchased from BCRCof FIRDI, accession number: BCRC 60001) was used. Macrophages RAW 264.7were placed in a 10 cm Petri dish having OMEN (containing 10% FBS, 4 mMglutamine, and 1.5 g/L sodium bicarbonate, pH=7.2˜7.4), and werecultivated in an incubator with culture conditions set at 37° C. and 5%CO₂.

When 80˜90% of the bottom area of the Petri dish was covered bymacrophages RAW 264.7, and cell growth patterns observed usingmicroscope were normal, DMEM was removed from the Petri dish. 3 mL offresh DMEM was added into the Petri dish. The fresh DMEM in the Petridish was sucked and discharged using a pipette so as to repeatedly rinsethe cells in the bottom of the Petri dish. Thus, the cells were detachedfrom the bottom of the Petri dish, and a suspension was formed. A properamount of the suspension was added into a new Petri dish containing 10mL of DMEM, followed by cultivation in an incubator with cultureconditions set at 37° C. and 5% CO₂.

B. Preparation of EPS Test Solution:

The EPS solution (845 mg/L) produced by Pediococcus acidilactici05B0111, which was obtained in the section entitled “E. Screening oflactic acid bacteria isolates having great EPS-producing ability” of theabove Example 1, was diluted using sterile water so that EPS testsolutions having final concentrations of 422.5 mg/L, 169 mg/L, and 84.5mg/L were made. A portion of the original EPS solution (845 mg/L) wasused as an EPS test solution as well. All of the EPS test solutions werefiltered using a membrane filter have a pore size of 0.22 μm for furtheruse.

C. Evaluation for Ability of EPS Produced by Pediococcus acidilactici05B0111 to Stimulate Proinflammatory Cytokine Secretion of MacrophageRAW 264.7:

Before performing this experiment, macrophages RAW 264.7 subjected tosub-culture according to procedures as described in the precedingsection, entitled “A. Source and cultivation of macrophage”, wereacquired, and cell concentration was adjusted using DMEM. Consequently,a cell suspension having a cell concentration of 8×10⁵ cell/mL was made.100 μL of the aforementioned cell suspension of macrophages RAW 264.7was added into each well of a 96-well plate, followed by centrifugationat 1000 rpm for 5 minutes. The 96-well plate was then placed in anincubator (37° C., 5% CO₂) for 1-2 hours for cultivation. Therefore,macrophages RAW 264.7 were attached to the bottom of each well.Macrophages RAW 264.7 were arranged into 7 groups that include 4experiment groups, 2 control groups (a positive group and a negativegroup), and 1 blank group. The 7 groups were added with the followingmaterials: (1) each of the experimental groups 1,2,3,4 was added with 20μL of the respective one of the EPS test solutions having theconcentrations of 84.5 mg/L, 169 mg/L, 422.5 mg/L, and 845 mg/L; (2) thepositive control group was added with 20 μL of lipopolysaccharide (LPS)(10 μg/mL); (3) the negative control group was added with 20 μL of DMEM;and (4) the blank group was added with 20 μL of sterile water.

After cultivation in an incubator (37° C., 5% CO₂) for 24 hours, theliquid in each well was obtained so as to conduct enzyme linkedimmunosorbent assay (ELISA) using mouse TNF-α/TNFSF1A (R&D Systems, Cat.No. DY410). Absorbances (OD₄₅₀) of the 4 experimental groups weremeasured at a wavelength of 450 nm, and were converted to concentrations(pg/mL) according to a correlation curve that was previously generatedby plotting concentrations of TNF-α standards and the respectiveabsorbances (OD₄₅₀). Additionally, a portion of the cell culture of theexperimental group 4 and a portion of the cell culture of the blankgroup were analyzed by virtue of Cytometric Bead Array using MouseInflammation Kit (BD, Cat. No. 552364).

D. Evaluation for Ability of EPS Produced by Pediococcus Acidilactici05B0111 to Stimulate Nitric Oxide Secretion of Macrophage RAW 264.7:

Macrophages RAW 264.7 subjected to sub-culture according to proceduresas described in the preceding section, entitled “A. Source andcultivation of macrophage”, were plated in each well (1×10⁵ cell/well)of a 96-well plate, followed by overnight cultivation at 37° C.Macrophages RAW 264.7 were arranged into four groups including threeexperiment groups and one positive control group. Each of the threeexperiment groups (designated as 1%, 5%, and 10% EPS groups) was addedwith 100 μL of a respective one of three solutions that all contain 100ng/mL lipopolysaccharide and 1 ng/mL IFN-γ, and that respectivelycontain 1% (v/v), 5% (v/v), and 10% (v/v) EPS test solutions. Thepositive control group was added with 100 μL of a solution containingonly 100 ng/mL lipopolysaccharide and 1 ng/mL IFN-γ. Cultivation wasconducted at 37° C. for 24 hours.

50 μL of the liquid in each of the wells was acquired and was added intoa respective well of a new 96-well plate for the following nitric oxideassay (NO assay). 50 μL of sulfanilamide (60 mM) and 50 μL ofN-1-naphthylethylenediamine (4 mM) were added into each of the wells ofthe aforementioned new 96-well plate, followed by shaking for 5 minutes.Absorbance (OD₅₄₀) of the mixture in each of the wells was measured at awavelength of 540 nm using the ELISA reader. OD₅₄₀ of the mixture wasconverted to a nitrite concentration according to a correlation curvethat was generated beforehand by plotting different known concentrationsof sodium nitrite (NaNO₂) and the respective absorbances (OD₅₄₀). Thenitrite concentration of the positive control group was regarded as100%, and relative percentage of nitrite concentrations of the three EPSgroups was calculated based on the nitrite concentration of the positivecontrol group.

Results:

The results of ELISA in the preceding section, entitled “C. Evaluationfor ability of EPS produced by Pediococcus acidilactici 05B0111 tostimulate proinflammatory cytokine secretion of macrophage RAW 264.7”,are shown in FIG. 4. Referring to FIG. 4, the concentration of TNF-αsecreted by macrophages RAW 264.7 increases with the increase of the EPSconcentration. The results show that EPS produced by Pediococcusacidilactici 05B0111 are capable of stimulating macrophages RAW 264.7 tosecrete TNF-α. Namely, EPS produced by Pediococcus acidilactici 05B0111are able to assist in activation of macrophages.

The results of analysis using Cytometric Bead Array are as follows.Regarding the blank group, after macrophages RAW 264.7 were cultured for24 hours using the sterile water, the concentrations of IL-6, IL-10,MCP-1, IFN-γ, TNF, and IL-12p70 were respectively 0 pg/mL, 0 pg/mL, 113pg/mL, 0 pg/mL, 1362 pg/mL, and 0 pg/mL. Regarding the experimentalgroup 4, after macrophages RAW 264.7 were cultured for 24 hours usingthe EPS test solution having the concentration of 845 mg/L, theconcentrations of IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70 wererespectively 7 pg/mL, 68 pg/mL, 215 pg/mL, 26 pg/mL, 2473 pg/mL, and4.92 pg/mL. The results indicate: EPS produced by Pediococcusacidilactici 05B0111 of this invention can stimulate macrophages RAW264.7 to secrete TNF, but are not capable of effectively stimulatingmacrophages RAW 264.7 to secrete IL-6, IL-10, MCP-1, IFN-γ, andIL-12p70.

The results of NO assay are as follows. After 24 hours of culture formacrophages RAW 264.7 using the respective one of the 1%, 5%, and 10%EPS test solutions, relative percentage of nitrite concentration of eachof the EPS groups is reduced. The degree of reduction of relativepercentage of nitrite concentration tends to be more obvious when EPSconcentration increases. Particularly, nitrite concentration of the 10%EPS group is reduced to about 69% of the nitrite concentration of thepositive control group. The results prove that EPS produced byPediococcus acidilactici 05B0111 can repress NO secretion of macrophagesRAW 264.7 and are hence able to alleviate inflammatory reactions.

TNF-α can be used as an index of activation of macrophages, andproinflammatory cytokines and NO can serve as indices of inflammatoryreactions. Therefore, based on the aforementioned results, theapplicants believe: EPS produced by Pediococcus acidilactici 05B0111 ofthis invention can activate macrophages, but do not stimulatemacrophages to induce inflammatory reactions, thereby being able tomodulate immune activity.

Example 7 Evaluation for Ability of EPS Produced by PediococcusAcidilactici 05B0111 Cultivated Using Different Carbohydrates as CarbonSources to Stimulate TNF-α Secretion of Mouse Macrophages ExperimentalMaterials and Procedures:

The inoculums of Pediococcus acidilactici 05B0111, which were obtainedin the section entitled “B. Preparation of inoculums of lactic acidbacteria isolates” of the above Example 1, were inoculated into each of15 mL of MRS broth (containing 10% glucose), 15 mL of MRS-sucrose broth,and 15 mL of MRS-maltose broth so that each of the MRS broth (designatedas the glucose group), the MRS-sucrose broth (designated as the sucrosegroup), and the MRS-maltose broth (designated as the maltose broth) hada final inoculum concentration of 1% (v/v). Cultivation was thenconducted at 37° C. for 72 hours. Isolation and purification of EPS inthe resultant culture, and determination of EPS concentration of theresultant culture were performed according to the procedures asdescribed in the sections, entitled “A. Isolation and purification ofexopolysaccharides” and “B. Determination of EPS concentration”, of theGeneral Experimental Procedures. The purified EPS were dissolved inddH₂O, followed by 2-fold, 5-fold, and 10-fold dilution using sterilewater. Therefore, EPS test solutions having different concentrationswere prepared. According to the procedures as described in the section,entitled “C. Evaluation for ability of EPS produced by Pediococcusacidilactici 05B0111 to stimulate proinflammatory cytokine secretion ofmacrophage RAW 264.7”, of the above Example 6, the EPS test solutionshaving different concentrations were used to stimulate macrophages RAW264.7 to secrete TNF-α, and the concentration of TNF-α secreted bymacrophages RAW 264.7 was measured. Macrophages RAW 264.7 that werecultured with sterile water served as the blank group.

Results:

The results are shown in Table 3.

TABLE 3 Evaluation for ability of EPS produced by Pediococcusacidilactici 05B0111 cultivated using different carbohydrates as carbonsources to stimulate TNF-α secretion of macrophagesRAW 264.7Concentration EPS test solution of TNF-α Group Concentration(mg/L)(pg/mL) Glucose 233.20 5145.04 116.60 1081.73 46.64 234.64 23.32 92.74Sucrose 845.11 2473.00 422.56 1358.94 169.02 1039.80 84.51 784.38Maltose 242.80 3810.41 121.40 892.49 48.56 239.42 24.28 74.32 Blank 0502

Referring to Table 3, EPS produced by Pediococcus acidilactici 05B0111using glucose, sucrose, and maltose as carbon sources are all capable ofstimulating macrophages RAW 264.7 to secrete TNF-α, and theconcentration of TNF-α secreted by macrophages RAW 264.7 increases withthe increase of EPS concentration. In addition, compared to the blankgroup, EPS of the glucose group, the sucrose group, and the maltosegroup have different abilities to stimulate TNF-α secretion ofmacrophages RAW 264.7 under the different EPS concentrations. Theapplicants deduce: molecular weight, structure, and degree of branchingregarding EPS produced by Pediococcus acidilactici 05B0111 might varywith carbon sources utilized by Pediococcus acidilactici 05B0111, andability of EPS to stimulate TNF-α secretion of macrophages might behence affected.

All patents and literature references cited in the present specificationas well as the references described therein, are hereby incorporated byreference in their entirety. In case of conflict, the presentdescription, including definitions, will prevail.

While the invention has been described with reference to the abovespecific embodiments, it is apparent that numerous modifications andvariations can be made without departing from the scope and spirit ofthis invention. It is therefore intended that this invention be limitedonly as indicated by the appended claims.

1. An isolated Pediococcus acidilactici 05B0111 capable of producing anexopolysaccharide, said Pediococcus acidilactici 05B0111 being depositedin Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)under an accession number DSM
 22345. 2. A method of producing anexopolysaccharide comprising cultivating an isolated Pediococcusacidilactici in a suitable medium under condition such that theexopolysaccharide is formed.
 3. The method of claim 2, wherein theisolated Pediococcus acidilactici is an isolated Pediococcusacidilactici 05B0111 capable of producing an exopolysaccharide, saidPediococcus acidilactici 05B0111 being deposited in Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH (DSMZ) under an accessionnumber DSM
 22345. 4. The method of claim 2, further comprising purifyingthe exopolysaccharide.
 5. The method of claim 2, wherein the suitablemedium comprises a carbon source selected from the group consisting of:lactose, fructose, maltose, glucose, molasses, galactose, xylose,xylitol, inulin, sorbitol, trehalose, sucrose, and mixtures thereof. 6.The method of claim 5, wherein the suitable medium is an ediblematerial.
 7. The method of claim 6, wherein the edible material isselected from the group consisting of: a fluid milk product, milkpowder, fruit juice, soybean milk, vegetable-fruit juice, health food,animal feed, an agricultural product, a livestock product, an aquaticproduct, and a functional ingredient.
 8. The method of claim 2, wherein,before cultivating, the suitable medium has a pH value ranging from 3 to7.
 9. The method of claim 8, wherein, before cultivating, the suitablemedium has a pH value of
 5. 10. The method of claim 2, wherein thecultivating step is conducted at a temperature ranging from 25° C. to37° C.
 11. A pharmaceutical composition comprising an isolatedPediococcus acidilactici 05B0111 of claim
 1. 12. The pharmaceuticalcomposition of claim 11, further comprising an exopolysaccharideobtained by cultivating an isolated Pediococcus acidilactici in asuitable medium under condition such that the exopolysaccharide isformed, wherein the isolated Pediococcus acidilactici is an isolatedPediococcus acidilactici 05B0111 capable of producing anexopolysaccharide, said Pediococcus acidilactici 05B0111 being depositedin Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)under an accession number DSM
 22345. 13. The pharmaceutical compositionof claim 11, further comprising a pharmaceutically acceptable carrier.14. A food product comprising an isolated Pediococcus acidilactici05B0111 of claim
 1. 15. The food product of claim 14, further comprisingan exopolysaccharide obtained by cultivating an isolated Pediococcusacidilactici in a suitable medium under condition such that theexopolysaccharide is formed, wherein the isolated Pediococcusacidilactici is an isolated Pediococcus acidilactici 05B0111 capable ofproducing an exopolysaccharide, said Pediococcus acidilactici 05B0111being deposited in Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ) under an accession number DSM 22345.